Terpenoids form the largest class of natural products, exhibiting astounding structural complexity and a correspondingly wide range of biological activity. While a few such compounds have found their way into clinical use, many more have been reported to exhibit promising pharmaceutical activity. Unfortunately, further investigation is often restricted by severe limitations in amount of available material. This hinders not only direct use, but also the semi-synthetic derivation approach that has proven so useful in generating clinically relevant compounds. Thus, there is a pressing need to provide general access to terpenoid natural products, yet the accompanying structural complexity prevents generally applicable synthetic routes. As the next step towards our long-term goal of engineering the production of targeted libraries and specific individual terpenoid `natural'products for pharmaceutical investigation and use, we propose to build on our success in modular metabolic engineering of bacterial diterpene production by expanding this modular approach to include the multiple downstream oxygenation reactions required for elaboration of the terpene olefin intermediates to bioactive terpenoid natural products. These oxygenation reactions are typically carried out by microsomal cytochromes P450 in partnership with an associated reductase, requiring extensive engineering to enable such `downstream'elaboration of terpenes to terpenoids. Based on our preliminary results indicating that the terpene olefin intermediate can be efficiently transferred from bacteria engineered for its production to co-cultured bacteria engineered for subsequent functionalization, we have developed an extensively modular approach to engineering the production of highly functionalized terpenoids. Development of this modular engineering system will enable facile investigation of combinatorial biosynthesis, whose potential is suggested by the substrate promiscuity exhibited by many microsomal P450s, as well as functional identification of novel biosynthetic enzymes. Accordingly, in addition to development of the proposed modular approach, and given our particular focus on diterpenoid metabolism, we will use this metabolic engineering system to functionally characterize diterpenoid biosynthetic enzymes from both native sources and proposed enzymatic engineering efforts designed to increase substrate promiscuity. We expect that establishment of the proposed generally applicable modular metabolic engineering system for facile assembly of extended biosynthetic pathways will dramatically increase access to the extremely large class of terpenoid natural products. PUBLIC HEALTH RELEVANCE: This project proposes development of a general method for genetically engineering bacteria to produce elaborate compounds from the enormous class of terpenoid natural products (>50,000 known). The resulting small molecules are potential pharmaceutical agents whose investigation and use has been hindered by the very limited quantities that are typically available from the relevant native producing organism. Bacterial production will provide an effective source for production of the larger amounts of these natural products necessary for detailed investigation of their promising biological activity.